1. Field of the Invention
The present invention relates to techniques for characterizing biological materials through analyzing laser-induced light emissions. More specifically, the present invention relates to a method and apparatus for characterizing biological materials by performing a time-resolved and wavelength-resolved analysis on laser-induced fluorescence emissions from the biological materials.
2. Related Art
Laser-induced emission spectroscopy, in particular, a laser-induced fluorescence spectroscopy (LIFS) has been extensively applied to complex biological systems to diagnose human diseases, such as tumors or atherosclerotic plaques in the medical field, and to analyze chemical or biochemical composition of organic matters in other fields. The increased interest in
LIFS can be attributed to its noninvasive approach to obtain both qualitative and quantitative information of a biological system in vivo. In comparison to other spectroscopic techniques, LIFS provides several advantages, such as wavelength tunability, narrow bandwidth excitation, directivity, and short pulses excitation. Furthermore, LIFS can selectively and efficiently excite the fluorophores in organic matter and greatly improves the fluorescence selectivity and detectability.
Typically during an LIFS process, a sample is excited with a short pulse of light of a predetermined wavelength and intensity, and a reemission profile is measured following the excitation using a fast photodetector. LIFS measurements can be categorized as either static (i.e., steady-state or time-integrated) or dynamic (time-resolved). Typically, steady-state techniques provide an “integral” spectrum over time, which contains such information as fluorescence emission intensity, spectral distribution, and polarization/anisotropy. However, while using the steady-state systems, the time-dependence of emission and the potential information contained therein are ignored.
On the other hand, time-resolved techniques allow real-time evolution of the laser-induced emission to be directly recorded, which was made possible by the availability of short (nanoseconds) and ultra-short (picoseconds) pulsed lasers, as well as advances in high-speed electronics. Because the light emission process occurs in a very short time interval after the stimulating event (e.g., fluorescence decay time is in the order of nanoseconds), the time-resolved measurement can provide rich information about molecular species and protein structures of the sample. For example, the time-resolved techniques permit “early” processes (typically the direct excitation of short-lived states or very rapid subsequent reactions) and “late” processes (typically from long-lived states, delayed excitation by persisting electron populations or by reactions which follow the original electron process) to be “separated” in the measured data.
More importantly, the time-resolved measurement can be complemented by spectral information in the laser-induced emission to reveal additional characteristics of a sample. Note that the time-resolved measurement only obtains an integrated effect from a wide range of wavelengths. To resolve the laser-induced emission into component wavelengths while still being able to perform time-resolved measurement, some existing LIFS techniques use a scanning monochromator to select wavelengths from the broadband emission one wavelength at a time, and to direct the selected wavelength component to the photodetector. However, to resolve another wavelength from the emission spectrum, the sample has to be excited again to produce another reemission, while the monochromator is tuned to select the new wavelength.
Unfortunately, these existing techniques can take a significant amount of time to resolve multiple spectral components from a wide band light emission. Although each wavelength component can be recorded in real-time, the transition time of using a monochromator to select another wavelength can take up to a few seconds, which becomes the limiting factor in performing real-time measurements. Furthermore, an overall measurement can take a large amount of time if a large number of stimulation locations on the sample have to be measured.
Hence, what is needed is a method and an apparatus that facilitates near real-time recording of both time-resolved and wavelength-resolved information from a light emission caused by a single excitation of a sample.